{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T12:24:13Z","timestamp":1784118253466,"version":"3.55.0"},"reference-count":47,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T00:00:00Z","timestamp":1754956800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003593","name":"Conselho Nacional de Desenvolvimento Cient\u00c3fico e Tecnol\u00c3\u00b3gico","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003593","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002322","name":"Coordena\u00c3\u00a7\u00c3\u00a3o de Aperfei\u00c3\u00a7oamento de Pessoal de N\u00c3vel Superior","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100002322","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004901","name":"Funda\u00c3\u00a7\u00c3\u00a3o de Amparo \u00c3\u00a0 Pesquisa do Estado de Minas Gerais","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004901","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005636","name":"Funda\u00c3\u00a7\u00c3\u00a3o Arthur Bernardes","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100005636","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>The CRISPR-Cas technology can be used to disable drug resistance genes. Since carbapenem resistance can be mediated by multiple resistance determinants, we here investigated the extent of re-sensizitation when targeting the <jats:italic>bla<\/jats:italic><jats:sub>KPC<\/jats:sub> carbapenemase gene and assessed possible effects on porins and efflux pumps. While full re-sensitization was achieved in a laboratory strain of <jats:italic>Escherichia coli<\/jats:italic> solely equipped with <jats:italic>bla<\/jats:italic><jats:sub>KPC<\/jats:sub>, resistance reduction in a clinical isolate of <jats:italic>Klebsiella michiganensis<\/jats:italic> was achieved in 63% of analyzed transformants, which was a consequence of plasmid copy number reduction and decreased <jats:italic>bla<\/jats:italic><jats:sub>KPC<\/jats:sub> gene expression. Damages in the Cas9, as well as alterations in carbapenem-resistance promoting genes including <jats:italic>ompK36<\/jats:italic> downregulation and mutations in the <jats:italic>acrB<\/jats:italic> gene were found, likely preventing more efficient re-sensitization. Hence, interference with a single resistance gene promoted the emergence of clonal variants that exhibit alterations in outer membrane proteins. Those bacterial countermeasures, however, were not sufficient to restore the original carbapenem-resistant phenotype.<\/jats:p>","DOI":"10.1371\/journal.pone.0328521","type":"journal-article","created":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T17:30:09Z","timestamp":1755019809000},"page":"e0328521","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["CRISPR-Cas9 targeting the blaKPC gene in a clinical isolate of Klebsiella michiganensis: Reduction of imipenem resistance and changes in genomic carbapenem resistance determinants"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0612-4808","authenticated-orcid":true,"given":"Thaysa Leite","family":"Tagliaferri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7157-8454","authenticated-orcid":true,"given":"Alex","family":"Kr\u00fcttgen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiago Ant\u00f4nio de Oliveira","family":"Mendes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5406-5470","authenticated-orcid":true,"given":"Simone","family":"Gon\u00e7alves dos Santos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hans-Peter","family":"Horz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,8,12]]},"reference":[{"key":"pone.0328521.ref001","doi-asserted-by":"crossref","DOI":"10.1093\/jac\/dkab353","article-title":"The role of new carbapenem combinations in the treatment of multidrug-resistant Gram-negative infections","volume":"76","author":"E Bouza","year":"2021","journal-title":"J Antimicrob Chemother"},{"issue":"1","key":"pone.0328521.ref002","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1038\/nrmicro3380","article-title":"Molecular mechanisms of antibiotic resistance","volume":"13","author":"JMA Blair","year":"2015","journal-title":"Nat Rev Microbiol"},{"issue":"9","key":"pone.0328521.ref003","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1016\/S1473-3099(13)70190-7","article-title":"Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases","volume":"13","author":"LS Munoz-Price","year":"2013","journal-title":"Lancet Infect Dis"},{"issue":"4","key":"pone.0328521.ref004","doi-asserted-by":"crossref","first-page":"1151","DOI":"10.1128\/AAC.45.4.1151-1161.2001","article-title":"Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae","volume":"45","author":"H Yigit","year":"2001","journal-title":"Antimicrob Agents Chemother"},{"issue":"4","key":"pone.0328521.ref005","doi-asserted-by":"crossref","DOI":"10.1128\/cmr.00008-23","article-title":"Klebsiella pneumoniae carbapenemase variants: the new threat to global public health","volume":"36","author":"L Ding","year":"2023","journal-title":"Clin Microbiol Rev"},{"issue":"2","key":"pone.0328521.ref006","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1093\/jac\/dkx415","article-title":"Identification and genomic characterization of a KPC-2-, NDM-1- and NDM-5-producing Klebsiella michiganensis isolate","volume":"73","author":"B Zheng","year":"2018","journal-title":"J Antimicrob Chemother"},{"key":"pone.0328521.ref007","doi-asserted-by":"crossref","first-page":"100516","DOI":"10.1016\/j.nmni.2019.100516","article-title":"First clinical case of KPC-3-producing Klebsiella michiganensis in Europe","volume":"29","author":"SN Seiffert","year":"2019","journal-title":"New Microbes New Infect"},{"key":"pone.0328521.ref008","unstructured":"World Health Organization. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. 2017."},{"issue":"11","key":"pone.0328521.ref009","doi-asserted-by":"crossref","first-page":"1146","DOI":"10.1038\/nbt.3043","article-title":"Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials","volume":"32","author":"D Bikard","year":"2014","journal-title":"Nat Biotechnol"},{"issue":"11","key":"pone.0328521.ref010","doi-asserted-by":"crossref","first-page":"1141","DOI":"10.1038\/nbt.3011","article-title":"Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases","volume":"32","author":"RJ Citorik","year":"2014","journal-title":"Nat Biotechnol"},{"issue":"23","key":"pone.0328521.ref011","doi-asserted-by":"crossref","first-page":"7267","DOI":"10.1073\/pnas.1500107112","article-title":"Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria","volume":"112","author":"I Yosef","year":"2015","journal-title":"Proc Natl Acad Sci USA"},{"key":"pone.0328521.ref012","doi-asserted-by":"crossref","first-page":"578","DOI":"10.3389\/fmicb.2020.00578","article-title":"Exploring the potential of CRISPR-Cas9 under challenging conditions: facing high-copy plasmids and counteracting beta-lactam resistance in clinical strains of enterobacteriaceae","volume":"11","author":"TL Tagliaferri","year":"2020","journal-title":"Front Microbiol"},{"issue":"2","key":"pone.0328521.ref013","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/s41587-023-01759-y","article-title":"Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice","volume":"42","author":"YE Gencay","year":"2024","journal-title":"Nat Biotechnol"},{"issue":"2","key":"pone.0328521.ref014","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1093\/jac\/dks387","article-title":"Characterization of IncN plasmids carrying bla CTX-M-1 and qnr genes in Escherichia coli and Salmonella from animals, the environment and humans","volume":"68","author":"M Dolejska","year":"2013","journal-title":"J Antimicrob Chemother"},{"key":"pone.0328521.ref015","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gkab1111","article-title":"PLSDB: advancing a comprehensive database of bacterial plasmids","volume":"50","author":"GP Schmartz","year":"2022","journal-title":"Nucleic Acids Res"},{"issue":"2","key":"pone.0328521.ref016","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1128\/AAC.00734-08","article-title":"Phenotypic and enzymatic comparative analysis of the novel KPC variant KPC-5 and its evolutionary variants, KPC-2 and KPC-4","volume":"53","author":"DJ Wolter","year":"2009","journal-title":"Antimicrob Agents Chemother"},{"issue":"2","key":"pone.0328521.ref017","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1111\/1574-6968.12273","article-title":"Promoter deletions of Klebsiella pneumoniae carbapenemase (KPC)-encoding genes (blaKPC -2) and efflux pump (AcrAB) on \u03b2-lactam susceptibility in KPC-producing Enterobacteriaceae","volume":"348","author":"GD Seecoomar","year":"2013","journal-title":"FEMS Microbiol Lett"},{"issue":"1842","key":"pone.0328521.ref018","doi-asserted-by":"crossref","DOI":"10.1098\/rstb.2020.0478","article-title":"Plasmid co-infection: linking biological mechanisms to ecological and evolutionary dynamics","volume":"377","author":"C Igler","year":"2021","journal-title":"Phil Trans R Soc B"},{"issue":"4","key":"pone.0328521.ref019","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1021\/acssynbio.7b00462","article-title":"High-level dCas9 expression induces abnormal cell morphology in Escherichia coli","volume":"7","author":"S Cho","year":"2018","journal-title":"ACS Synth Biol"},{"key":"pone.0328521.ref020","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1099\/jmm.0.012575-0","article-title":"Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae","volume":"58","author":"D Landman","year":"2009","journal-title":"J Med Microbiol"},{"issue":"6","key":"pone.0328521.ref021","doi-asserted-by":"crossref","first-page":"1510","DOI":"10.1093\/jac\/dkw028","article-title":"Inactivation or inhibition of AcrAB-TolC increases resistance of carbapenemase-producing Enterobacteriaceae to carbapenems","volume":"71","author":"HTH Saw","year":"2016","journal-title":"J Antimicrob Chemother"},{"issue":"6","key":"pone.0328521.ref022","doi-asserted-by":"crossref","first-page":"1472","DOI":"10.1038\/ismej.2016.202","article-title":"The fitness costs and trade-off shapes associated with the exclusion of nine antibiotics by OmpF porin channels","volume":"11","author":"K Phan","year":"2017","journal-title":"ISME J"},{"issue":"3","key":"pone.0328521.ref023","doi-asserted-by":"crossref","DOI":"10.1371\/journal.ppat.1007218","article-title":"Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen","volume":"15","author":"A Fajardo-Lubi\u00e1n","year":"2019","journal-title":"PLoS Pathog"},{"issue":"12","key":"pone.0328521.ref024","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1038\/nrmicro1994","article-title":"The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria","volume":"6","author":"J-M Pag\u00e8s","year":"2008","journal-title":"Nat Rev Microbiol"},{"issue":"3","key":"pone.0328521.ref025","doi-asserted-by":"crossref","first-page":"103901","DOI":"10.1016\/j.resmic.2021.103901","article-title":"Analysis of AcrB in Klebsiella pneumoniae reveals natural variants promoting enhanced multidrug resistance","volume":"173","author":"Y Li","year":"2022","journal-title":"Res Microbiol"},{"issue":"2","key":"pone.0328521.ref026","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1128\/CMR.00117-14","article-title":"The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria","volume":"28","author":"X-Z Li","year":"2015","journal-title":"Clin Microbiol Rev"},{"issue":"5","key":"pone.0328521.ref027","doi-asserted-by":"crossref","first-page":"1188","DOI":"10.1093\/jac\/dkv475","article-title":"Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli","volume":"71","author":"M Adler","year":"2016","journal-title":"J Antimicrob Chemother"},{"issue":"2","key":"pone.0328521.ref028","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1111\/mmi.14162","article-title":"Protease-deficient SOS constitutive cells have RecN-dependent cell division phenotypes","volume":"111","author":"AR Warr","year":"2019","journal-title":"Mol Microbiol"},{"issue":"2","key":"pone.0328521.ref029","doi-asserted-by":"crossref","first-page":"106721","DOI":"10.1016\/j.ijantimicag.2023.106721","article-title":"RecA inactivation as a strategy to reverse the heteroresistance phenomenon in clinical isolates of Escherichia coli","volume":"61","author":"S Diaz-Diaz","year":"2023","journal-title":"Int J Antimicrob Agents"},{"key":"pone.0328521.ref030","doi-asserted-by":"crossref","first-page":"100229","DOI":"10.1016\/j.focha.2023.100229","article-title":"Potential of the endogenous and artificially inserted CRISPR-Cas system for controlling virulence and antimicrobial resistance of food pathogens","volume":"2","author":"RC Rodrigues","year":"2023","journal-title":"Food Chem Adv"},{"key":"pone.0328521.ref031","unstructured":"Registry of standard biological parts. plasmid backbones\/assembly. 2019 [Accessed 2018 September 10]. http:\/\/parts.igem.org\/Plasmid_backbones\/Assembly"},{"issue":"6","key":"pone.0328521.ref032","article-title":"A comparison and optimization of methods and factors affecting the transformation of Escherichia coli","volume":"33","author":"W-T Chan","year":"2013","journal-title":"Bioscience Reports"},{"issue":"1","key":"pone.0328521.ref033","first-page":"000748","article-title":"ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes","volume":"8","author":"AF Florensa","year":"2022","journal-title":"Microb Genom"},{"key":"pone.0328521.ref034","volume-title":"Performance Standards for Antimicrobial Susceptibility Testing","author":"Clinical and Laboratory Standards Institute","year":"2019","edition":"27"},{"issue":"10","key":"pone.0328521.ref035","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1093\/bioinformatics\/btu048","article-title":"Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases","volume":"30","author":"S Bae","year":"2014","journal-title":"Bioinformatics"},{"issue":"10","key":"pone.0328521.ref036","doi-asserted-by":"crossref","first-page":"3779","DOI":"10.1128\/JB.01861-07","article-title":"Quantitative analysis of three hydrogenotrophic microbial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic bacteria, within plaque biofilms associated with human periodontal disease","volume":"190","author":"ME Vianna","year":"2008","journal-title":"J Bacteriol"},{"issue":"2","key":"pone.0328521.ref037","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0057923","article-title":"The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses","volume":"8","author":"T V\u011btrovsk\u00fd","year":"2013","journal-title":"PLoS One"},{"issue":"4","key":"pone.0328521.ref038","doi-asserted-by":"crossref","first-page":"1328","DOI":"10.1128\/AEM.66.4.1328-1333.2000","article-title":"rRNA operon copy number reflects ecological strategies of bacteria","volume":"66","author":"JA Klappenbach","year":"2000","journal-title":"Appl Environ Microbiol"},{"issue":"1","key":"pone.0328521.ref039","doi-asserted-by":"crossref","DOI":"10.1186\/1471-2105-6-62","article-title":"A standard curve based method for relative real time PCR data processing","volume":"6","author":"A Larionov","year":"2005","journal-title":"BMC Bioinform"},{"issue":"3","key":"pone.0328521.ref040","doi-asserted-by":"crossref","first-page":"90","DOI":"10.7171\/jbt.12-2303-004","article-title":"SeqTrace: a graphical tool for rapidly processing DNA sequencing chromatograms","volume":"23","author":"BJ Stucky","year":"2012","journal-title":"J Biomol Tech"},{"key":"pone.0328521.ref041","unstructured":"Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010. http:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc"},{"issue":"15","key":"pone.0328521.ref042","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: a flexible trimmer for Illumina sequence data","volume":"30","author":"AM Bolger","year":"2014","journal-title":"Bioinformatics"},{"issue":"5","key":"pone.0328521.ref043","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing","volume":"19","author":"A Bankevich","year":"2012","journal-title":"J Computat Biol"},{"issue":"14","key":"pone.0328521.ref044","doi-asserted-by":"crossref","first-page":"2068","DOI":"10.1093\/bioinformatics\/btu153","article-title":"Prokka: rapid prokaryotic genome annotation","volume":"30","author":"T Seemann","year":"2014","journal-title":"Bioinformatics"},{"key":"pone.0328521.ref045","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gky1050","article-title":"PLSDB: a resource of complete bacterial plasmids","volume":"47","author":"V Galata","year":"2019","journal-title":"Nucleic Acids Res"},{"issue":"4","key":"pone.0328521.ref046","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pcbi.1006946","article-title":"TASmania: a bacterial toxin-antitoxin systems database","volume":"15","author":"H Akarsu","year":"2019","journal-title":"PLoS Comput Biol"},{"issue":"10","key":"pone.0328521.ref047","doi-asserted-by":"crossref","first-page":"3396","DOI":"10.1128\/AAC.00285-06","article-title":"High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35\/36 insertional inactivation, and down-regulation of the phosphate transport porin phoe","volume":"50","author":"FM Kaczmarek","year":"2006","journal-title":"Antimicrob Agents Chemother"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0328521","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T17:30:16Z","timestamp":1755019816000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0328521"}},"subtitle":[],"editor":[{"given":"Yung-Fu","family":"Chang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,8,12]]},"references-count":47,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8,12]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0328521","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,12]]}}}